PREreview of HIV-1 uncoating by release of viral cDNA from capsid-like structures in the nucleus of infected cells

The authors of the manuscript seek to better understand the mechanism by which HIV-1 uncoats capsid (CA) and integrates its own viral cDNA into the host genome. They hypothesize that the entire viral capsid along with the integration complex is required for proper viral integration. They accomplish this by infecting cells with VSV-G psuedotyped virus to deliver a viral particle that can be visualized from infection up until integration. Using live cell imaging, they show that largely complete HIV-1 capsid enters the nucleus of the cell and where then the viral cDNA is released by physical disruption of the capsid lattice. These findings corroborate with other very recent publications using structural techniques that show HIV-1 capsid partially disassembles via the HIV-1 integration complex. Moreover, the authors show that the mechanism is also conserved in primary cells which further strengthens the paper.

Overall, the manuscript seeks to answer a very important question in the HIV field that remains poorly understood and is important for a clearer understanding of the viral lifecycle. The manuscript is well written and proposes an important hypothesis with results that clearly test it. By using an innovative viral delivery system, they were able to visualize the process of viral uncoating and integration, however, it could be strengthened by using a lower multiplicity of infection that is closer to an in vivo infection. The data contribute to the field’s understanding of important HIV-1 biology and also brings about new questions about the viral lifecycle, such as the mechanism behind how the integration complex physically ruptures the viral capsid.

As a whole, the manuscript itself is strong in its current state. The authors successfully test the hypothesis they set out to investigate and do so in a manner that provides further questions. The manuscript is publishable in its current state with minor revisions.    

The only major concern for the manuscript is that recently published structural studies have not been included in the citations and should be. I have a number of other constructive suggestions that may help to strengthen the paper.

Major Comments/Essential Revisions:

·     In figure 2, a similar experimental setup from the first figure is used, however, the time point is changed to 24 hours without much reasoning. Using the same time point throughout the manuscript would provide a stronger foundation for experimental results. Or, perhaps, the authors can provide an explanation about why the time point was changed.

·     Figure 3 has an issue with a lot of background for IN SNAP.SiR imaging. The high amount of background for this marker should be cleaned up in order to properly assess the images and localization. This problem also persists in the quantification methods for this figure. Foci per cell were used to quantify this marker, however, in its current state it should not be. This was not considered a major problem because of the in vivo data shown later in the paper. However, the paper would be strengthened by improving the signal to background in this figure, if possible. If not, perhaps this should be mentioned as a caveat of the quantification.

·     Tomographic slices depicted in figure 4 are very difficult to discern. This could be improved by providing more slices or a clearer averaged image.  If this is not possible, perhaps this panel could be moved to the supplementary material because it does not add much. The 3-D reconstructed image is beautiful and provides stronger support for the authors’ claims.  

·     Figure 5 would perhaps benefit from reducing the number of viral components that are present in the experiment. There are several very bright foci and the images themselves are difficult to distinguish cellular components. These images could perhaps be improved by reducing the viral components to a minimum as well as using several slices or clearer images. Or, at least, the EM image and the fluorescent photomicrographs could be shown individually as well as superimposed.

Minor Comments:

·     Related to the last point above, throughout the manuscript, a MOI of 6 is used and results in several punctae on a per cell basis. Lowering the MOI to result in less punctae per cell could help better visualize the experiments and provide clearer pictures.

·     Figure 4 very nicely depicts the colocalization of several markers in the nucleus, however, it suffers from the same problems as previous figures where there are too many viral markers on a per cell basis. The experimental setup could benefit from reducing the number of viral components to the minimum required for visualization.

·     The final figure itself is very strong in that the images are much clearer to interpret because there are only a couple of foci per cell. The images themselves reproduce what was observed in previous figures but are in primary cells. These images could be improved by trying to clean up background, however, these images are how the authors should aim to represent images in the other figures.